Computational protocol: HIV-1 escapes from N332-directed antibody neutralization in an elite neutralizer by envelope glycoprotein elongation and introduction of unusual disulfide bonds

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Protocol publication

[…] Proviral env genes from PBMCs that were infected in vitro with a single clonal HIV-1 variant were PCR-amplified and sequenced [–]. Nucleotide sequences were aligned using ClustalW in the software package of BioEdit [], and edited manually, excluding contamination. The gp160 sequences were used to construct a Maximum Likelihood (ML) tree. The best-fit nucleotide substitution model (GTR + I+G), selected by hierarchical likelihood ratio tests (hLRTs, Model Test 3.7 []) was implemented in the heuristic search for the best ML tree applying the Tree Bisection and Reconnection (TBR) branch-swapping algorithm using PAUP*4.0 [], starting with a neighbor-joining (NJ) tree constructed under the Hasegawa–Kishino–Yano (HKY85) model of evolution []. The robustness of the NJ phylogeny was assessed by bootstrap analysis with 1000 rounds of replication.Genetic analyses were performed on gp160 sequences starting at nucleotide position 91, and for gp160 protein analyses starting at amino acid residue 31, thereby excluding the signal peptide. PNGS, and NXT and NXS motifs were identified using N-glycosite [] at the Los Alamos HIV database website (http://www.hiv.lanl.gov/content/sequence/GLYCOSITE/glycosite.html). Overlapping PNGS (NN[TS][ST]) were included by N-glycosite as one PNGS, and NPS or NPT motifs were excluded. […]

Pipeline specifications

Software tools Clustal W, BioEdit, PAUP*
Application Phylogenetics
Organisms Human immunodeficiency virus 1
Chemicals Amino Acids, Cysteine